Patent US5930119A successfully refreshed from Google Patents.
EP0510693A2
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Expression of human serum albumin in pichia pastoris
Assignee: Research Corp Technologies Inc
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Filing: 1992-04-24
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Granted: —
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Expires: 2012-04-24
Abstract
Abstract A novel expression cassette, vectors and process for the secretion of HSA in Pichia pastoris cells. In accordance with the present invention there has been discovered an improved expression cassette for the production of HSA in Pichia pastoris comprising a) a Pichia pastoris 5' regulatory region having a 5' end and a 3' end selected from the group consisting of the Pichia pastoris AOX1 5' regulatory region and the Pichia pastoris DAS1 5' regulatory region wherein the 3' end of the 5' regulatory region is operably linked to; b) an HSA structural gene encoding a signal sequence and mature protein having a 5' end and a 3' end wherein the HSA structural gene has an ATG start codon within about 11 deoxyribonucleotides of the 5' end of said HSA structural gene; and operably linked to c) a 3' termination sequence. <IMAGE>
Inventors
- 👤 Kotikanyad Sreekrishna
- 👤 Kathryn Ann Barr
- 👤 Russell A. Brierley
- 👤 Gregory P. Thill
- 👤 Juerg F. Tschopp
Bibliographic Details
Application Number
—
Jurisdiction
EP
Data Source
google_patents
Last Synchronized
2026-11-09 22:02
CPC Classifications
C; C12; C12N; C12N15/00; C12N15/09; C12N15/63; C12N15/79; C12N15/80; C12N15/81; C12N15/815; C07; C07K; C07K14/00; C07K14/435; C07K14/76; C07K14/765
Claims Analysis
Total Claims: 12
Claim 1
Independent Claim
114 words
An expression cassette for the production of HSA in Pichia pastoris comprising a) a Pichia pastoris 5' regulatory region having a 5' end and a 3' end selected from a Pichia pastoris AOX1 5' regulatory region and a Pichia pastoris DAS1 5' regulatory region wherein the 3' end of the 5' regulatory region is operably linked to b) a HSA structural gene encoding a HSA signal sequence and a mature HSA protein, having a 5' end and a 3' end wherein the HSA structural gene has an ATG start codon within no more than 11 deoxyribonucleotides of the 5' end of said HSA structural gene; operably linked to c) a suitable 3' termination sequence.
Claim 2
Dependent Claim
29 words
The expression cassette of claim 1 wherein the 5' regulatory region is selected from the AOX1 5' regulatory region and the DAS1 5' regulatory region from Pichia pastoris .
Claim 3
Dependent Claim
33 words
The expression cassette of claim 1 wherein the 3' termination sequence is isolated from a Pichia pastoris gene selected from an AOX1 gene, a DAS1 gene, a p40 gene and a HIS4 gene.
Claim 4
Dependent Claim
29 words
The expression cassette of claim 1 wherein the expression cassette is incorporated into a vector selected from circular plasmids and linear plasmids, the latter preferably being integrative site-specific vectors.
Claim 5
Dependent Claim
93 words
The expression cassette of claim 1 wherein the expression cassette is incorporated into a vector comprising the following serial arrangement: a) a first insertable DNA fragment; b) at least one marker gene; and, c) a second insertable DNA fragment; wherein at least one expression cassette is incorporated either before or after the marker gene of component (b), and the first and second insertable DNA fragments employed are homologous with separate portions of the Pichia pastoris genome and the insertable fragments are in the same relative orientation as exist in the Pichia pastoris genome.
Claim 6
Dependent Claim
50 words
The expression cassette of claim 5 contained in said vector wherein the first insertable DNA fragment and the second insertable DNA fragment are obtained from the DNA sequences of a gene isolated from Pichia pastoris selected from an AOX1 gene, a p40 gene, a DAS1 gene and an HIS4 gene.
Claim 7
Dependent Claim
49 words
The expression cassette of claim 5 contained in said vector wherein the marker gene is selected from a Pichia pastoris HIS4 gene, a Pichia pastoris ARG4 gene, a Saccharomyces cerevisiae SUC2 gene, a G418 R gene of bacterial transposon Tn601 and a G418 R gene of bacterial transposon Tn903.
Claim 8
Dependent Claim
142 words
The expression cassette of claim 5 wherein said vector comprises a) a first insertable DNA fragment which is an operable 5' regulatory region from the AOX1 gene being about one kilobase in length isolated from Pichia pastoris operably linked to b) a HSA structural gene encoding a HSA signal sequence and a mature HSA protein having a 5' end and a 3' end wherein the HSA structural gene has a ATG start codon within no more than 8 deoxyribonucleotides of the 5' end of said HSA structural gene; operably linked to c) the 3' termination sequence of the AOX1 gene isolated from Pichia pastoris ; operably linked to d) a marker gene which is the HIS4 gene isolated from Pichia pastoris ; operably linked to e) a second insertable DNA fragment which is about 0.65 kilobases of the AOX1 3' termination sequence.
Claim 9
Dependent Claim
30 words
The expression cassette of claim 8 wherein the HSA structural gene has a ATG start codon with either the deoxyribonucleotide AGGAATTC or no deoxyribonucleotide 5' of said ATG start codon.
Claim 10
Dependent Claim
17 words
A Pichia pastoris cell transformed with an expression cassette according to any of claims 1 to 9.
Claim 11
Dependent Claim
56 words
The Pichia pastoris cell of claim 10 wherein the Pichia pastoris cell to be transformed is selected from Pichia pastoris GS115 (NRRL Y-15851), Pichia pastoris GS190 (NRRL Y-18014), Pichia pastoris PPF1 (NRRL Y-18017), Pichia pastoris (NRRL Y-11430) and Pichia pastoris (NRRL Y-11431), wherein Pichia pastoris GS 115 (NRRL Y-15851) is the most preferred Pichia pastoris cell.
Claim 12
Dependent Claim
62 words
A process for the secretion of HSA from transformed Pichia pastoris cells according to claim 10 or 11 comprising a) transforming a Pichia pastoris cell with at least one vector having at least one expression cassette according to any of claims 1 to 9, and b) culturing the resulting transformed Pichia pastoris cell under suitable conditions to obtain the production of HSA.
References Cited (Backward Citations)
No citation records indexed.
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